Background. Rodents could act as reservoir for Cryptosporidium spp. specially C. parvum, a zoonotic agent responsible for human infections. Since there is no information about Cryptosporidium infection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization of Cryptosporidium spp. in this region. Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method using SspI and VspI restriction enzymes was carried out to genotype the species and then obtained results were sequenced. Results. Three out of 100 samples were diagnosed as positive and overall prevalence of Cryptosporidium spp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples and C. parvum pattern was identified. Finally PCR-RFLP findings were sequenced and presence of C. parvum was confirmed again. Conclusions. Our study showed rodents could be potential reservoir for C. parvum. So an integrated program for control and combat with them should be adopted and continued.